Herbert W Virgin

National Institute of Allergy and Infectious Diseases, Maryland, United States

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Publications (184)1746.41 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: The essential immediate-early transcriptional activator RTA, encoded by gene 50, is conserved among all characterized gammaherpesviruses. Analyses of a recombinant murine gamma-herpesvirus 68 lacking both of the known gene 50 promoters (G50pDblKO) revealed that this mutant retained the ability to replicate in the simian kidney epithelial cell line Vero, but not in permissive murine fibroblasts following low MOI infection. However, G50pDblKo replication in permissive fibroblasts was partially rescued by high MOI infection. In addition, replication of the G50pDblKO virus was rescued by growth on MEFs isolated from IFNα/βR(-/-) mice, while growth on Vero cells was suppressed by the addition of IFNα. 5' rapid amplification of cDNA ends (RACE) analyses of RNA prepared from G50pDblKo and wild-type MHV68 infected murine macrophages identified three novel gene 50 transcripts initiating from 2 transcription initiation sites located upstream of the currently defined proximal and distal gene 50 promoters. In transient promoter assays neither of the newly identified gene 50 promoters exhibited sensitivity to IFNα treatment, although RTA levels were lower in IFNα-responsive cells infected with the G50pDblKo mutant. Infection of mice with the MHV68 G50pDblKo virus demonstrated that this mutant virus was able to establish latency in the spleen and peritoneal exudates cells (PECs) of C57Bl/6 mice with about 1/10 the efficiency of wild-type virus or marker rescue virus. However, despite the ability to establish latency, the G50pDblKo virus mutant was severely impaired in its ability to reactivate from either latently infected splenocytes or PECs. Consistent with the ability to rescue replication of the G50pDblKO mutant by growth on type I interferon receptor null MEFs, infection of IFNα/βR(-/-) mice with the G50pDblKo mutant virus demonstrated partial rescue of: (i) acute virus replication in the lungs; (ii) establishment of latency; and (iii) reactivation from latency. The identification of additional gene 50/RTA transcripts highlight the complex mechanisms involved in controlling expression of RTA, likely reflecting time dependent and/or cell-specific roles of different gene 50 promoters in controlling virus replication. Furthermore, the newly identified gene 50 transcripts may also act as negative regulators that modulate RTA expression. The viral transcription factor RTA, encoded by open reading frame 50 (Orf50), is well conserved among all known gammaherpesviruses and is essential for both virus replication and reactivation from latently infected cells. Previous studies have shown that regulation of gene 50 transcription is complex. The studies reported here describe the presence of additional alternatively initiated, spliced transcripts that encode RTA. Understanding how expression of this essential viral gene product is regulated may identify new strategies for interfering with infection in the setting of gammaherpesvirus-induced diseases.
    Journal of Virology 02/2014; · 5.08 Impact Factor
  • Jason M. Norman, Scott A. Handley, Herbert W. Virgin
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    ABSTRACT: Advanced sequencing techniques have shown that bacteria are not the only complex and important microbes in the human intestine. Non-bacterial organisms, particularly the virome and the mycobiome, are important regulators of intestinal immunity and inflammation. The virome is mucosal and systemic; it can alter the host response to bacteria and interact with host genes and bacteria to contribute to disease pathogenesis. The human mycobiome is also complex and can contribute to intestinal inflammation. We review what has recently been learned about the non-bacterial and non-archaeal microbes in the gastrointestinal tract, discussing their potential effects on health and disease and analytical approaches for their study. Studies of associations between the microbiome and intestinal pathology should incorporate kingdom-agnostic approaches if we are to fully understand intestinal health and disease.
    Gastroenterology 01/2014; · 12.82 Impact Factor
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    ABSTRACT: ABSTRACT Pervasive transcription is observed in a wide range of organisms, including humans, mice, and viruses, but the functional significance of the resulting transcripts remains uncertain. Current genetic approaches are often limited by their emphasis on protein-coding open reading frames (ORFs). We previously identified extensive pervasive transcription from the murine gammaherpesvirus 68 (MHV68) genome outside known ORFs and antisense to known genes (termed expressed genomic regions [EGRs]). Similar antisense transcripts have been identified in many other herpesviruses, including Kaposi's sarcoma-associated herpesvirus and human and murine cytomegalovirus. Despite their prevalence, whether these RNAs have any functional importance in the viral life cycle is unknown, and one interpretation is that these are merely "noise" generated by functionally unimportant transcriptional events. To determine whether pervasive transcription of a herpesvirus genome generates RNA molecules that are functionally important, we used a strand-specific functional approach to target transcripts from thirteen EGRs in MHV68. We found that targeting transcripts from six EGRs reduced viral protein expression, proving that pervasive transcription can generate functionally important RNAs. We characterized transcripts emanating from EGRs 26 and 27 in detail using several methods, including RNA sequencing, and identified several novel polyadenylated transcripts that were enriched in the nuclei of infected cells. These data provide the first evidence of the functional importance of regions of pervasive transcription emanating from MHV68 EGRs. Therefore, studies utilizing mutation of a herpesvirus genome must account for possible effects on RNAs generated by pervasive transcription. IMPORTANCE The fact that pervasive transcription produces functionally important RNAs has profound implications for design and interpretation of genetic studies in herpesviruses, since such studies often involve mutating both strands of the genome. This is a common potential problem; for example, a conservative estimate is that there are an additional 73,000 nucleotides transcribed antisense to annotated ORFs from the 119,450-bp MHV68 genome. Recognizing the importance of considering the function of each strand of the viral genome independently, we used strand-specific approaches to identify six regions of the genome encoding transcripts that promoted viral protein expression. For two of these regions, we mapped novel transcripts and determined that targeting transcripts from these regions reduced viral replication and the expression of other viral genes. This is the first description of a function for these RNAs and suggests that novel transcripts emanating from regions of pervasive transcription are critical for the viral life cycle.
    mBio 01/2014; 5(2). · 5.62 Impact Factor
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    ABSTRACT: Autophagy is a key innate immune response to intracellular parasites that promotes their delivery to degradative lysosomes following detection in the cytosol or within damaged vacuoles. Like Listeria and Shigella, which use specific mechanisms to avoid autophagic detection and capture, the bacterial pathogen Francisella tularensis proliferates within the cytosol of macrophages without demonstrable control by autophagy. To examine how Francisella evades autophagy, we screened a library of F. tularensis subsp. tularensis Schu S4 HimarFT transposon mutants in GFP-LC3-expressing murine macrophages by microscopy for clones localised within autophagic vacuoles after phagosomal escape. Eleven clones showed autophagic capture at six hours post-infection, whose HimarFT insertions clustered to four genetic loci involved in lipopolysaccharidic and capsular O-antigen biosynthesis. Consistent with the HimarFT mutants, in-frame deletion mutants of two representative loci, FTT1236 and FTT1448c (manC), lacking both LPS and capsular O-antigen, underwent phagosomal escape but were cleared from the host cytosol. Unlike wild type Francisella, the O-antigen deletion mutants were ubiquitinated, and recruited the autophagy adaptor p62/SQSTM1 and LC3 prior to cytosolic clearance. Autophagy-deficient macrophages partially supported replication of both mutants, indicating that O-antigen-lacking Francisella are controlled by autophagy. These data demonstrate the intracellular protective role of this bacterial surface polysaccharide against autophagy.
    Cellular Microbiology 11/2013; · 4.81 Impact Factor
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    ABSTRACT: The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.
    Nature 11/2013; · 38.60 Impact Factor
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    ABSTRACT: Compact viral genomes such as those found in noroviruses, which cause significant enteric disease in humans, often encode only a few proteins, but affect a wide range of processes in their hosts and ensure efficient propagation of the virus. Both human and mouse noroviruses persistently replicate and are shed in stool, a highly effective strategy for spreading between hosts. For mouse norovirus (MNV), the presence of a glutamate rather than an aspartate at position 94 of the NS1/2 protein was previously shown to be essential for persistent replication and shedding. Here, we analyze these critical sequences of NS1/2 at the structural level. Using solution NMR methods we determined folded NS1/2 domain structures from a nonpersistent murine norovirus strain CW3, a persistent strain CR6, and a persistent mutant strain CW3(D94E) . We found an unstructured PEST-like domain followed by a novel folded domain in the N-terminus of NS1/2. All three forms of the domain are stable and monomeric in solution. Residue 94, critical for determining persistence, is located in a reverse turn following an α-helix in the folded domain. The longer sidechain of glutamate, but not aspartate, allows interaction with the indole group of the nearby tryptophan, reshaping the surface of the domain. The discrimination between glutamyl and aspartyl residue is imposed by the stable tertiary conformation. These structural requirements correlate with the in vivo function of NS1/2 in persistence, a key element of norovirus biology and infection. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
    Proteins Structure Function and Bioinformatics 11/2013; · 3.34 Impact Factor
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    ABSTRACT: Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.
    The Journal of molecular diagnostics: JMD 11/2013; · 3.48 Impact Factor
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    ABSTRACT: Delivery of granule contents to epithelial surfaces by secretory cells is a critical physiologic process. In the intestine, goblet cells secrete mucus that is required for homeostasis. Autophagy proteins are required for secretion in some cases, though the mechanism and cell biological basis for this requirement remain unknown. We found that in colonic goblet cells, proteins involved in initiation and elongation of autophagosomes were required for efficient mucus secretion. The autophagy protein LC3 localized to intracellular multi-vesicular vacuoles that were consistent with a fusion of autophagosomes and endosomes. Using cultured intestinal epithelial cells, we found that NADPH oxidases localized to and enhanced the formation of these LC3-positive vacuoles. Both autophagy proteins and endosome formation were required for maximal production of reactive oxygen species (ROS) derived from NADPH oxidases. Importantly, generation of ROS was critical to control mucin granule accumulation in colonic goblet cells. Thus, autophagy proteins can control secretory function through ROS, which is in part generated by LC3-positive vacuole-associated NADPH oxidases. These findings provide a novel mechanism by which autophagy proteins can control secretion.
    The EMBO Journal 11/2013; · 9.82 Impact Factor
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    ABSTRACT: Previous studies identified a role for latent herpesvirus infection in cross-protection to infection and exacerbation of chronic inflammatory diseases. Here, we identified over 600 genes differentially expressed in spleens, livers, or brains of mice latently infected with gammaherpesvirus 68 and found distinct sets of genes linked to different pathways were altered in spleen compared to liver. Several of the most differentially expressed latency-specific genes (e.g. IFNγ, Cxcl9, Ccl5) are associated with known latency-specific phenotypes.
    Journal of Virology 10/2013; · 5.08 Impact Factor
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    ABSTRACT: & Aims: Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting anti-microbial peptides (AMPs). Autophagy is an important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice. We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1(f/f) x Villin-cre) or CD11c(+) cells (Atg16l1(f/f) x CD11c-cre); these mice were used to assess cell type-specific, anti-bacterial autophagy. All responses were compared to Atg16l1(f/f) mice (controls). Mice were infected with Salmonella enterica serovar Typhimurium; cecum and small intestine tissues were collected for immunofluorescence, histology, and quantitative reverse transcription PCR analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on anti-bacterial responses in human epithelial cells. Autophagy was induced in small intestine and cecum following infection with S Typhimurium, and required Atg16l1. S Typhimurium colocalized with microtubule-associated protein 1 light chain 3 beta (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1(f/f) x Villin-cre mice. Atg16l1(f/f) x Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMP. Consistent with these defective immune responses, Atg16l1(f/f) x Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1(f/f) x CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1. Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It is also required to prevent systemic infection of mice with enteric bacteria.
    Gastroenterology 08/2013; · 12.82 Impact Factor
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    ABSTRACT: Lethal viral infections produce widespread inflammation with vascular leak, clotting and bleeding (disseminated intravascular coagulation, termed DIC), organ failure, and high mortality. Serine proteases in clot-forming (thrombotic) and clot-dissolving (thrombolytic) cascades are activated by an inflammatory cytokine storm and also can induce systemic inflammation with loss of normal serine protease inhibitor (serpin) regulation. Myxomavirus secretes a potent anti-inflammatory serpin, Serp-1, that inhibits clotting factor X (fX) and thrombolytic tissue- and urokinase-type plasminogen activators (tPA, uPA) with anti-inflammatory activity in multiple animal models. Purified serpin significantly improved survival in a murine Gammaherpesvirus-68 (MHV68) infection in interferon gamma receptor (IFNγR) knock-out mice, a model for lethal inflammatory vascultis. Treatment of MHV68 infected mice with neuroserpin, a mammalian serpin that inhibits only tPA and uPA, was ineffective. Serp-1 reduced virus load, lung hemorrhage, and aortic, lung, and colon inflammation in MHV68 infected mice and also reduced virus load. Neuroserpin suppressed a wide range of immune spleen cell responses after MHV68 infection, while Serp-1 selectively increased CD11c+ splenocytes (macrophage and dendritic cells) and reduced CD11b+ tissue macrophage. Serp-1 altered gene expression for coagulation and inflammatory responses whereas neuroserpin did not. Serp-1 treatment was assessed in a second viral infection, mouse-adapted Zaire Ebolavirus in wild type BALB/c mice, with improved survival and reduced tissue necrosis. In summary, treatment with this unique myxomavirus-derived serpin suppresses systemic serine protease and innate immune responses caused by unrelated lethal viral infections (both RNA and DNA viruses), providing a potential new therapeutic approach for treatment of lethal viral sepsis.
    Antimicrobial Agents and Chemotherapy 06/2013; · 4.57 Impact Factor
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    ABSTRACT: [This corrects the article on p. e1003118 in vol. 9.].
    PLoS Pathogens 05/2013; 9(5). · 8.14 Impact Factor
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    ABSTRACT: Norovirus (NV) gastroenteritis is a major contributor to global morbidity and mortality, yet little is known about immune mechanisms leading to NV control. Previous studies using the murine norovirus (MNV) model have established a key role for T cells in MNV clearance. Despite these advances, important questions remain regarding the magnitude, location, and dynamics of the MNV-specific T cell response. To address these questions, we identified MNV-specific MHC class I immunodominant epitopes using an overlapping peptide screen. One of these epitopes (ORF2(519-527)) was highly conserved among all NV genogroups. Using MHC class I/peptide tetramers, we tracked MNV-specific CD8 T cells in lymphoid and mucosal sites during infection with two MNV strains with distinct biological behavior: acutely-cleared CW3 and persistent CR6. Here, we show that enteric MNV infection elicited robust T cell responses primarily in the intestinal mucosa, and that MNV-specific CD8 T cells dynamically regulated the expression of surface molecules associated with activation, differentiation, and homing. Furthermore, compared to MNV-CW3, chronic infection with MNV-CR6 resulted in fewer and less functional CD8 T cells, and this difference was evident as early as day 8 post-infection. Finally, MNV-specific CD8 T cells were capable of reducing viral load in persistently infected Rag1(-/-) mice, suggesting that these cells are a crucial component of NV immunity. Collectively, these data provide fundamental new insights into the adaptive immune response to two closely-related NV strains with distinct biological behavior and bring us closer to understanding the correlates of protective antiviral immunity in the intestine.
    Journal of Virology 04/2013; · 5.08 Impact Factor
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    ABSTRACT: IFN-γ activates cells to restrict intracellular pathogens by upregulating cellular effectors including the p65 family of guanylate-binding proteins (GBPs). Here we test the role of Gbp1 in the IFN-γ-dependent control of T. gondii in the mouse model. Virulent strains of T. gondii avoided recruitment of Gbp1 to the parasitophorous vacuole in a strain-dependent manner that was mediated by the parasite virulence factors ROP18, an active serine/threonine kinase, and the pseudokinase ROP5. Increased recruitment of Gbp1 to Δrop18 or Δrop5 parasites was associated with clearance in IFN-γ-activated macrophages in vitro, a process dependent on the autophagy protein Atg5. The increased susceptibility of Δrop18 mutants in IFN-γ-activated macrophages was reverted in Gbp1(-/-) cells, and decreased virulence of this mutant was compensated in Gbp1(-/-) mice, which were also more susceptible to challenge with type II strain parasites of intermediate virulence. These findings demonstrate that Gbp1 plays an important role in the IFN-γ-dependent, cell-autonomous control of toxoplasmosis and predict a broader role for this protein in host defense.
    PLoS Pathogens 04/2013; 9(4):e1003320. · 8.14 Impact Factor
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    ABSTRACT: The lysosomal degradation pathway of autophagy has a crucial role in defence against infection, neurodegenerative disorders, cancer and ageing. Accordingly, agents that induce autophagy may have broad therapeutic applications. One approach to developing such agents is to exploit autophagy manipulation strategies used by microbial virulence factors. Here we show that a peptide, Tat–beclin 1—derived from a region of the autophagy protein, beclin 1, which binds human immunodeficiency virus (HIV)-1 Nef—is a potent inducer of autophagy, and interacts with a newly identified negative regulator of autophagy, GAPR-1 (also called GLIPR2). Tat–beclin 1 decreases the accumulation of polyglutamine expansion protein aggregates and the replication of several pathogens (including HIV-1) in vitro, and reduces mortality in mice infected with chikungunya or West Nile virus. Thus, through the characterization of a domain of beclin 1 that interacts with HIV-1 Nef, we have developed an autophagy-inducing peptide that has potential efficacy in the treatment of human diseases. Autophagy functions in metazoans in cellular and tissue homeostasis, physiology, development, and protection against disease, and abnor-malities in autophagy may contribute to many different pathophysio-logical conditions 1,2 . Thus, strategies that augment autophagy may prevent or treat human disease 3 . Although some drugs in clinical use are capable of augmenting autophagy, these compounds exert pleiotropic effects, revealing an unmet need to develop specific indu-cers of autophagy. We sought to develop a specific autophagy-inducing agent with a potentially wide range of therapeutic effects. As viruses often provide key insights into the functionally important domains of host proteins, we investigated the molecular determinants governing the interaction between beclin 1, an essential autophagy protein in the class III phos-phatidylinositol-3-OH kinase (PI(3)K) complex involved in auto-phagic vesicle nucleation 4 , and the HIV-1 virulence factor, Nef 5 . These investigations led us to identify a Nef-interacting sequence of beclin 1 that is necessary and sufficient for autophagy induction and which provided the basis for the development of an autophagy-indu-cing peptide drug that has benefits in the clearance of polyglutamine expansion protein aggregates and the treatment of infectious diseases.
    · 38.60 Impact Factor
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    ABSTRACT: Autophagosome formation is a dynamic process that is strictly controlled by autophagy-related (Atg) proteins. However, how these Atg proteins are recruited to the autophagosome formation site or autophagic membranes remains poorly understood. Here, we found that FIP200, which is involved in proximal events, directly interacts with Atg16L1, one of the downstream Atg factors, in an Atg14- and phosphatidylinositol 3-kinase-independent manner. Atg16L1 deletion mutants, which lack the FIP200-interacting domain, are defective in proper membrane targeting. Thus, FIP200 regulates not only early events but also late events of autophagosome formation through direct interaction with Atg16L1.
    EMBO Reports 02/2013; · 7.19 Impact Factor
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    ABSTRACT: The lysosomal degradation pathway of autophagy has a crucial role in defence against infection, neurodegenerative disorders, cancer and ageing. Accordingly, agents that induce autophagy may have broad therapeutic applications. One approach to developing such agents is to exploit autophagy manipulation strategies used by microbial virulence factors. Here we show that a peptide, Tat-beclin 1-derived from a region of the autophagy protein, beclin 1, which binds human immunodeficiency virus (HIV)-1 Nef-is a potent inducer of autophagy, and interacts with a newly identified negative regulator of autophagy, GAPR-1 (also called GLIPR2). Tat-beclin 1 decreases the accumulation of polyglutamine expansion protein aggregates and the replication of several pathogens (including HIV-1) in vitro, and reduces mortality in mice infected with chikungunya or West Nile virus. Thus, through the characterization of a domain of beclin 1 that interacts with HIV-1 Nef, we have developed an autophagy-inducing peptide that has potential efficacy in the treatment of human diseases.
    Nature 01/2013; · 38.60 Impact Factor
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    ABSTRACT: Autophagy is a conserved homeostatic process in which cytoplasmic contents are degraded and recycled. Two ubiquitin-like conjugation pathways are required for the generation of autophagosomes, and ATG5 is necessary for both of these processes. Studies in mice deficient in ATG5 reveal a key role for autophagy in T lymphocyte function, as well as in B cell development and B-1a B cell maintenance. However, the role of autophagy genes in B cell function and antibody production has not been described. Using mice in which Atg5 is conditionally deleted in B lymphocytes, we showed here that this autophagy gene is essential for plasma cell homeostasis. In the absence of B cell ATG5 expression, antibody responses were significantly diminished during antigen-specific immunization, parasitic infection and mucosal inflammation. Atg5-deficient B cells maintained the ability to produce immunoglobulin and undergo class-switch recombination, yet had impaired SDC1 expression, significantly decreased antibody secretion in response to toll-like receptor ligands, and an inability to upregulate plasma cell transcription factors. These results build upon previous data demonstrating a role for ATG5 in early B cell development, illustrating its importance in late B cell activation and subsequent plasma cell differentiation.
    Autophagy 01/2013; 9(4). · 12.04 Impact Factor
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    ABSTRACT: The use of NextGen Sequencing clinically necessitates the need for informatics tools that support the complete workflow from sample accessioning to data analysis and reporting. To address this need we have developed Clinical Genomicist Workstation (CGW). CGW is a secure, n-tiered application where web browser submits requests to application servers that persist the data in a relational database. CGW is used by Washington University Genomic and Pathology Services for clinical genomic testing of many cancers. CGW has been used to accession, analyze and sign out over 409 cases since November, 2011. There are 22 ordering oncologists and 7 clinical genomicists that use the CGW. In summary, CGW a 'soup-to-nuts' solution to track, analyze, interpret, and report clinical genomic diagnostic tests.
    AMIA Summits on Translational Science proceedings AMIA Summit on Translational Science. 01/2013; 2013:156-157.
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    ABSTRACT: Although the transcription factors IRF-3 and IRF-7 are considered master regulators of type I interferon (IFN) induction and IFN stimulated gene (ISG) expression, Irf3(-/-)×Irf7(-/-) double knockout (DKO) myeloid dendritic cells (mDC) produce relatively normal levels of IFN-β after viral infection. We generated Irf3(-/-)×Irf5(-/-)×Irf7(-/-) triple knockout (TKO) mice to test whether IRF-5 was the source of the residual induction of IFN-β and ISGs in mDCs. In pathogenesis studies with two unrelated positive-sense RNA viruses (West Nile virus (WNV) and murine norovirus), TKO mice succumbed at rates greater than DKO mice and equal to or approaching those of mice lacking the type I IFN receptor (Ifnar(-/-)). In ex vivo studies, after WNV infection or exposure to Toll-like receptor agonists, TKO mDCs failed to produce IFN-β or express ISGs. In contrast, this response was sustained in TKO macrophages following WNV infection. To define IRF-regulated gene signatures, we performed microarray analysis on WNV-infected mDC from wild type (WT), DKO, TKO, or Ifnar(-/-) mice, as well as from mice lacking the RIG-I like receptor adaptor protein MAVS. Whereas the gene induction pattern in DKO mDC was similar to WT cells, remarkably, almost no ISG induction was detected in TKO or Mavs(-/-) mDC. The relative equivalence of TKO and Mavs(-/-) responses suggested that MAVS dominantly regulates ISG induction in mDC. Moreover, we showed that MAVS-dependent induction of ISGs can occur through an IRF-5-dependent yet IRF-3 and IRF-7-independent pathway. Our results establish IRF-3, -5, and -7 as the key transcription factors responsible for mediating the type I IFN and ISG response in mDC during WNV infection and suggest a novel signaling link between MAVS and IRF-5.
    PLoS Pathogens 01/2013; 9(1):e1003118. · 8.14 Impact Factor

Publication Stats

9k Citations
1,746.41 Total Impact Points

Institutions

  • 2012–2013
    • National Institute of Allergy and Infectious Diseases
      Maryland, United States
    • Donald Danforth Plant Science Center
      San Luis, Missouri, United States
  • 2007–2013
    • University of Texas Southwestern Medical Center
      • Department of Microbiology
      Dallas, Texas, United States
    • San Diego State University
      • Department of Biology
      San Diego, California, United States
  • 1995–2013
    • Washington University in St. Louis
      • • Midwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research (MRCE)
      • • Department of Pathology and Immunology
      San Luis, Missouri, United States
  • 1993–2012
    • University of Washington Seattle
      • • Department of Immunology
      • • Department of Pathology
      • • Department of Medicine
      Seattle, WA, United States
  • 2009–2011
    • University of Texas Medical Branch at Galveston
      • Department of Pathology
      Galveston, Texas, United States
    • Centers for Disease Control and Prevention
      • Division of Viral Diseases
      Atlanta, MI, United States
  • 2008
    • Mount Sinai School of Medicine
      • Department of Microbiology
      Manhattan, NY, United States
    • University of North Carolina at Chapel Hill
      • Department of Microbiology and Immunology
      North Carolina, United States
    • The Scripps Research Institute
      La Jolla, California, United States
  • 2006
    • Hospital of the University of Pennsylvania
      • Department of Pathology and Laboratory Medicine
      Philadelphia, Pennsylvania, United States
    • State University of New York Upstate Medical University
      • Department of Microbiology and Immunology
      Syracuse, New York, United States
  • 2002–2003
    • Emory University
      • Department of Microbiology and Immunology
      Atlanta, GA, United States
  • 1993–2003
    • University of Colorado
      • • Department of Microbiology
      • • Department of Neurology
      Denver, CO, United States
  • 1998
    • Eastern Virginia Medical School
      • Department of Microbiology and Molecular Cell Biology
      Norfolk, Virginia, United States
  • 1996
    • University of Western Australia
      Perth City, Western Australia, Australia
  • 1985–1992
    • Harvard Medical School
      • • Department of Microbiology and Immunobiology
      • • Department of Pathology
      Boston, MA, United States